The present invention relates generally to methods and compositions for the purification of CD34+ cells and specific antigens. The invention is more particularly directed to methods and compositions useful for releasing CD34+ cells, or compounds having a specific carbohydrate epitope of the CD34 surface antigen from a binding partner, which may be immobilized on an affinity matrix. The invention is also directed to methods for identifying compounds that bind to CD34+ cells.
Bone marrow transplantation is a critical procedure for treatment of a variety of diseases, such as leukemia, cancer and certain genetic diseases. The transplantation process involves the repopulation of a patient""s bone marrow stem cells following ablative treatment such as chemotherapy or radiation therapy. Serious problems can result, however, from transplantation of impure preparations of bone marrow stem cells. In allogenic transplantations (i e., transplantation with bone marrow cells from a genetically different donor), preparations that contain other types of cells. particularly T lymphocytes, can induce graft vs. host disease in the patient. This problem can be prevented through the use of autologous transplantation (i.e., transplantation with the patient""s own marrow). However, preparations of the patient""s marrow cells may be contaminated with diseased cells, resulting in ineffective treatment.
To alleviate the problems associated with contaminated preparations, a variety of methods for purification of stem cells are currently employed. Such methods often employ affinity procedures to selectively remove specific contaminating cells from a stem cell preparation. For example, monoclonal antibodies to a variety of contaminating cells types, such as T cells and cancer cells, have been employed. Because of the wide variety of contaminating cells, however, purification procedures based on selective removal of individual cell types are often incomplete. Such procedures also may involve many steps, rendering the purification process extremely costly and inefficient.
The purification process may also use affinity procedures to selectively isolate the desired cells. For example, monoclonal antibodies have been employed that selectively recognize an epitope on the CD34 antigen. Such antibodies may be used to isolate cells expressing the CD34 antigen, which include nearly all stem cells and early stage committed B lymphoid cells. These antibodies do not bind mature B cells, T cells, NK cells, monocytes, granulocytes, platelets or erythrocytes. Accordingly, the use of such specific antibodies can provide a more effective, and lower cost, method of purifying bone marrow stem cells.
However, while CD34-specific affinity matrices containing immobilized antibodies have significant advantages in the capture of cells expressing the CD34 antigen, the release of CD34+ cells from such affinity matrices has been problematic. Release is commonly effected by physically shearing the cells from the matrix. This procedure can cause cell damage, and can leave antibody bound to the released cells. Alternatively, enzymatic cleavage may be employed, to degrade the antibodv to which the CD34 antigen is bound. This procedure has the advantage of decreasing cell damage and removing bound antibody from the cells. However, the enzymes emploved could damage other cell surface proteins, and are inconvenient to use because of the necessity to monitor cell viability after treatment.
Accordingly, there is a need in the art for a method for releasing CD34+ cells from affinity matrices that overcomes the disadvantages encountered with existing methods. The present invention fulfills this need and provides further related advantages.
Briefly stated, this invention provides methods and compositions for the release of CD34+ cells, and specific surface antigens thereof, from affinity matrices. In one aspect of the invention, methods are provided for releasing a CD34+ cell from a CD34+ -binding partner complex, comprising contacting a complex of a CD34+ cell and a binding partner with a carbohydrate having the structure Neu5Acxcex12-3Galxcex21-4(X), wherein (X) is GlcNAc or a structurally similar analog thereof. In a preferred embodiment, the carbohydrate has the structure Neu5Acxcex12-3(Galxcex21-4GlcNAcxcex21-3)n, wherein n is an integer from 1 to 50.
In a related aspect of the invention, methods are provided for purifying CD34+ cells, comprising the steps of: (a) contacting a biological sample containing CD34+ cells with a binding partner, to form a CD34+ -binding partner complex; (b) separating the CD34+ -binding partner complex from unbound biological sample; (c) contacting the CD34+ -binding partner complex with a carbohydrate having the structure Neu5Acxcex12-3Galxcex21-4(X), wherein (X) is GlcNAc or a structurally similar analog thereof; and (d) collecting the CD34+ cells.
In yet another aspect of the present invention, methods are provided for releasing a molecule that binds monoclonal antibody 12.8 from a binding partner, comprising contacting a complex of the molecule and a binding partner with a carbohydrate having the following structure Neu5Acxcex12-3Galxcex21-4(X), wherein (X) is GlcNAc or a structurally similar analog thereof.
In a related aspect, this invention provides methods for purifying a molecule that binds monoclonal antibody 12.8, comprising the steps of: (a) contacting a sample containing a molecule that binds monoclonal antibody 12.8 with a binding partner, to form a complex; (b) separating the complex from unbound sample; (c) contacting the complex with a carbohydrate having the structure Neu5Acxcex12-3Galxcex21-4(X), wherein (X) is GlcNAc or a structurally similar analog thereof; and (d) collecting the molecule.
In still another aspect of this invention, methods are provided for screening for a candidate compound able to bind to CD34+ cells, comprising the steps of: (a) contacting a candidate compound with a carbohydrate having the structure Neu5Acxcex12-3Galxcex21-4(X), wherein (X) is GlcNAc or a structurally similar analog thereof; and (b) detecting candidate compound bound to the carbohydrate, thereby identifying a candidate compound that binds to CD34+ cells.
These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.